Death from cancer is often due to metastases arising from heterogeneous disseminated cancer cells that comprise therapy resistant cell clones. To overcome selection of variant cells by adjuvant therapies it is therefore necessary to search for and target common mutations shared by all disseminated cancer cells. Most likely only early mutations that occur prior to dissemination fulfill this postulate and are transmitted to all cells eventually found throughout the organism. In an attempt to define DNA changes shared among the progenitor cells of metastasis, we isolated and characterized single disseminated cancer cells from the bone marrow of breast cancer patients. To identify genomic regions affected by early DNA damage we therefore developed a protocol for array-based CGH of single cells. The novel technique should enable the detection of numerical imbalances in single cells and thereby increases cellular resolution of array / matrix-CGH at least 1.000 fold. Various methods of array preparation were compared with standard array-CGH. Additionally, oligo-arrays were tested and were not suited for single-cell DNA in our hands. While large amounts of unamplified DNA were suited for all standard arrays, chromosomal aberrations could not be retrieved when single-cell DNA was hybridized. We therefore developed an optimized protocol for BAC preparation, single-cell DNA-labeling, and hybridization and applied it successfully to single cells. We tested single normal cells from healthy donors, single leukocytes from patients with trisomy 21 and single disseminated tumor cells from bone marrow of breast cancer patients. While single normal cells displayed a normal karyotype, copy number ratios of 3:2 for chromosome 21 were readily detected in single leukocytes from a patient with trisomy 21. The novel 3K single cell BAC array with a resolution of 1Mb now enables whole-genome characterization of early progenitor cells of metachronic metastasis.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]